Molecular basis of antibiotic multiresistance transfer in Staphylococcus aureus

Jonathan S. Edwardsa,1, Laurie Bettsb, Monica L. Fraziera,2, Rebecca M. Polleta, Stephen M. Kwongc, William G. Waltonb, W. Keith Ballentine IIIb, Julianne J. Huangb, Sohrab Habibib, Mark Del Campod, Jordan L. Meiere, Peter. B. Dervane, Neville Firthc, and Matthew R. Redinboa,b,3

Departments of aBiochemistry and Biophysics and bChemistry, University of North Carolina, Chapel Hill, NC 27599; cSchool of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia; dRigaku Americas Corporation, The Woodlands, TX 77381; and eDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91106

Edited by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved December 21, 2012 (received for review November 12, 2012)

Multidrug-resistant Staphylococcus aureus infections pose a signif- relaxases, like those from the F and R388 , which con- icant threat to human health. Antibiotic resistance is most com- tain more than one catalytic (7). The C-terminal region monly propagated by conjugative plasmids like pLW1043, the first (residues 221–665) of NES is unique to pLW1043 and related S. vancomycin-resistant S. aureus vector identified in humans. We aureus plasmids, and its function has remained unclear. Most present the molecular basis for resistance transmission by the - relaxases are fused to catalytic C-terminal domains like a ing in S. aureus (NES), which is essential for conjugative (e.g., R1162) or (e.g., F, R388) that are essential ’ transfer. NES initiates and terminates the transfer of plasmids that to DNA transfer. In contrast, pLW1043 s NES C-terminal region variously confer resistance to a range of drugs, including vanco- shares no sequence similarity with other bacterial proteins (13). mycin, gentamicin, and mupirocin. The NES N-terminal relaxase– Here, we present crystal structures of the N- and C-terminal – domains of NES, as well as a small-angle X-ray scattering DNA complex crystal structure reveals unique protein DNA con- – tacts essential in vitro and for conjugation in S. aureus. Using this (SAXS) structure of the full-length NES DNA complex. We structural information, we designed a DNA minor groove-targeted show the two domains of NES work in concert to process DNA, polyamide that inhibits NES with low micromolar efficacy. The design a minor-groove targeted polyamide that inhibits NES at low micromolar concentrations, and establish specific regions of crystal structure of the 341-residue C-terminal region outlines a NES essential for plasmid transfer in S. aureus. Together, these unique architecture; in vitro and cell-based studies further estab- data unravel the molecular basis for antibiotic resistance acqui- lish that it is essential for conjugation and regulates the activity of sition by S. aureus and suggest potential new avenues for dis- the N-terminal relaxase. This conclusion is supported by a small- rupting the spread of multidrug resistance. angle X-ray scattering structure of a full-length, 665-residue NES– DNA complex. Together, these data reveal the structural basis for Results antibiotic multiresistance acquisition by S. aureus and suggest NES Crystal Structures. The pLW1043 nes gene was subcloned into novel strategies for therapeutic intervention. Escherichia coli expression vectors used to produce full-length, as well as N-terminal and C-terminal forms of NES. The crystal MRSA | USA300 | F-plasmid | pSK41 | hairpin structure of the NES relaxase (residues 2–195) was determined in complex with a 30-nt DNA sequence to 2.9 Å resolution (Fig. ntibiotic resistance, which arises in bacterial pathogens 1A and Table S1). The NES relaxase exhibits a structure similar Athrough conjugative plasmid DNA transfer, is a well-estab- overall to that observed for the other conjugative relaxases re- lished threat to global health. For example, whereas vancomycin solved to date (sharing 2.2, 2.9, 3.1, and 3.1 Å Cα rmsd with has been essential in treating recalcitrant Staphylococcus aureus the MobA, F, R388, and pCU1 relaxases, respectively) (15–18) infections for decades, vancomycin-resistant S. aureus (VRSA) and contains a metal ion coordinated by three and an strains have now appeared in clinical settings worldwide (1–3). adjacent catalytic tyrosine 25 (mutated to phenylalanine in this VRSA first arose in the United States through the interplay of structure) (Fig. 1A). Mass spectrometry data indicate that the conjugative DNA transfer and resistance-determinant trans- bound metal ion is nickel (Table S2), which is one of the ions position. The resulting plasmid, pLW1043, has been sequenced that we show supports NES relaxase activity in vitro (Fig. S2). and contains not only a vanHAX vancomycin-resistance trans- The bound DNA oligonucleotide contains a 7-bp duplex hairpin poson, but also a cadre of putative DNA transfer genes (4). It followed by a 9-nt ordered single-stranded region that extends was recently shown that the S. aureus plasmid pSK41, which is into the relaxase’s . closely related to pLW1043, mediates the transfer of vancomycin resistance from Enterococcus faecalis into strains of methicillin resistant S. aureus (MRSA) (5). Conjugative bacterial plasmids Author contributions: J.S.E., L.B., M.D.C., J.L.M., P.B.D., N.F., and M.R.R. designed research; use almost exclusively plasmid-encoded factors that work in J.S.E., L.B., M.L.F., R.M.P., S.M.K., W.G.W., W.K.B., J.J.H., S.H., M.D.C., J.L.M., and N.F. concert to coordinate the cell-to-cell transfer of one strand of performed research; J.S.E., L.B., M.L.F., R.M.P., S.M.K., W.G.W., W.K.B., J.J.H., S.H., the duplex plasmid (6, 7). An element common to all conjugative M.D.C., J.L.M., P.B.D., N.F., and M.R.R. analyzed data; and J.S.E., L.B., R.M.P., M.D.C., processes is the plasmid-encoded relaxase enzyme that initiates J.L.M., P.B.D., N.F., and M.R.R. wrote the paper. and terminates transfer by creating a transient single-strand DNA The authors declare no conflict of interest. break and covalent protein–DNA intermediate (8, 9). This article is a PNAS Direct Submission. The vancomycin-resistance plasmid pLW1043 (4) and related Database deposition: The atomic coordinates and structure factors have been deposited plasmids from S. aureus (10, 11), including pSK41 and pGO1 in the Protein Data Bank, www.pdb.org (PDB ID codes 4HT4 and 4HTE). – (12 14) as well as plasmids from streptococcal, lactococcal, and 1Present address: Department of Biological Engineering, Massachusetts Institute of Tech- clostridial strains, encode a relaxase enzyme termed nicking nology, Cambridge, MA 02139. Staphylococcus 2 enzyme in (NES) that exhibits a unique full- Present address: Laboratory of Molecular , National Institute of Environmental length sequence (Fig. S1). It is 665 residues in length, confines its Health Sciences Research, National Institutes of Health, Research Triangle Park, relaxase motifs to its N-terminal ∼220 aa, and encodes a “single NC 27709. tyrosine”-type relaxase like MobA from plasmid R1162 (15), 3To whom correspondence should be addressed. E-mail: [email protected]. although the NES and MobA relaxases share only 28% sequence This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. identity. NES is distinct from the second major class of conjugative 1073/pnas.1219701110/-/DCSupplemental.

2804–2809 | PNAS | February 19, 2013 | vol. 110 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1219701110 Downloaded by guest on September 26, 2021 NES relaxase outlined above (Fig. 1A). In the structure, 12 nt contact 21 residues of the NES relaxase, forming 17 base-specific interactions and 9 nonspecific phosphate interactions (Fig. 2A). Of particular note, DNA hairpin-binding loop 1 (residues 78–82) interacts with the DNA hairpin duplex minor groove and forms two base-specific interactions and one phosphate contact (Fig. 2B). DNA hairpin-binding loop 2 works in concert with loop 1 to close around the DNA duplex (Figs. 1A and 2A). Loop 2 (resi- dues 150–156) binds to the major groove of the hairpin, forming 6 base-specific interactions and 4 phosphate contacts (Fig. 2C). The single-stranded nine ordered nucleotides of the oriTHP30 oligonucleotide form 12 base-specific and 2 nonspecific phos- phate interactions with the relaxase (Fig. 2D). The nucleotide base of guanine 26 is buried in a cavity created by several res- idues, including two making hydrogen-bonding contacts. Fluo- rescence anisotropy polarization studies show that eliminating this region of the oligonucleotide reduces relaxase binding by 20-fold (oriT27 vs. oriT24(-3); Table S3). Thus, NES employs specific major- and minor-groove contacts around the DNA hair- pin duplex, as well as backbone and unique base-specificinter- actions, to align the DNA substrate for .

NES Catalysis. DNA cleavage assays using a 5′-6-FAM–labeled substrate (oriTHP37; Table S4) revealed the NES relaxase domain (residues 1–220) produces an equilibrium level of 15% (± 1%) cleaved DNA product (Fig. 3A), a value similar to that of relaxases examined previously (18, 19). In contrast to the related S. aureus pGO1 plasmid, which was reported to have a cleavage ∧ ∧ site at GCC C (where indicates the putative nic site) (14), we ∧ found that pLW1043 NES cleaves 2 nt upstream, at G CCC (where residues in boldface type indicate the validated nic site; Fig. S3 and Tables S3 and S4). NES relaxase mutants were then designed to test the role that specific regions of the enzyme play in catalytic function (Fig. 3A). An E86A substitution was found to reduce the cleavage-religation equilibrium significantly; in con- trast, a K22A mutation increases cleavage-religation equilibrium, whereas the addition of an adjacent charge (Ala-21-Glu) has no impact on the enzyme (Fig. 3A). The K22A and A21E mutant proteins show wild-type DNA binding; the E86A variant, however, exhibits only nonspecific binding, indicating the important role coordinated metal binding plays in substrate association (Table S5 and Fig. 2). K22 may interact with the DNA region downstream of the cleavage site, as its mutation to an alanine produces an equi- librium shift toward cleaved product (Fig. 3A; see also Fig. 2D). The elimination of DNA-binding loop 1 or 2 exerted dramatic impacts on NES activity. Removal of either of these regions shifted the cleavage-religation equilibrium of the enzyme to ∼90% cleaved DNA, close to trapping the enzyme in its covalent pro- tein–DNA complex state (Fig. 3A). This occurs despite the fact Fig. 1. NES structure from S. aureus pLW1043. (A) Crystal structure of the that the loop 1 or loop 2 deletion mutants exhibit either non- NES N-terminal relaxase (1–195) bound to a 30-nt oriT-containing hairpin specific or poor DNA binding relative to that in wild-type NES (OriTHP30). (B) Crystal structure of the C-terminal domain (254–593) with (Table S5). Similarly, changes in specific DNA nucleotides also domains highlighted in different shades of magenta. shift the NES cleavage-religation equilibrium toward cleavage, particularly the elimination of Gua-26 (Fig. 3B). Again, this occurs despite the impact these changes have on DNA binding. The crystal structure of the pLW1043 NES C-terminal domain The shifts in cleavage-religation equilibrium are reminiscent of (residues 254–593) was determined to 3.0 Å resolution and specific mutants as well as topoisomerase-targeted exhibits an all α-helical fold (α1–α16) with no structural homo- drugs that stabilize the covalent enzyme–DNA complex (20–22), logs identified using the Dali matrix-alignment method (Fig. 1B and they establish that loop 1, loop 2, and the Gua-26 binding and Table S1). Furthermore, it bears no structural similarity to pocket play important roles in NES relaxase catalysis. a helicase or a primase, like other relaxase-fused C-terminal do- The isolated NES relaxase (residues 1–220) exhibited no DNA mains examined previously. Together these structures provide cleavage until 4 nt were present past the nic site (oriTHP32; Fig. BIOCHEMISTRY unique atomic-level information on NES, an enzyme that drives 3C). After that, the stepwise addition of nucleotides produced a drug resistance propagation in S. aureus. stepwise increase in 1–220 relaxase activity up to 35% product formation (oriTHP33-45;Fig.3C). Intriguingly, when the full-length DNA Binding. Fluorescence anisotropy studies established that NES enzyme (1–665) was used, product equilibrium does not DNA oligonucleotides ∼30 nt in length containing duplex hairpin exceed ∼10% regardless of the length of oligonucleotide assayed bound the NES relaxase domain with affinities as low as 4 nM (Fig. 3C). Thus, the presence of the C-terminal domain signifi- (Tables S3 and S4). From this set, oligonucleotide oriTHP30 cantly impacts the catalytic activity of the N-terminal relaxase. (30 nt, with the origin of transfer, “oriT”, and a hairpin, “HP”; Furthermore, full-length NES is able to cleave DNA substrates 8nMaffinity, Table S3) produced the cocrystal structure with the with only 2- to 3-nt 3′ extensions, in contrast to 1–220 NES (Fig.

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Fig. 2. DNA binding by S. aureus NES. (A) Schematic of the NES relaxase–DNA contacts with bases examined using variant oligos in boldface type. (B)Minor groove-binding hairpin loop 1 of NES. (C) Major groove-binding hairpin loop 2 of NES. (D) Active site region of NES, with the buried Gua26 base highlighted.

3C). Taken together, these data indicate that the unique C-ter- by which the C-terminal and N-terminal regions work in concert minal domain of NES regulates the relaxase cleavage-religation to regulate the DNA cleavage-religation equilibrium of NES. equilibrium of the intact enzyme. Regions Essential for DNA Transfer in S. aureus. Conjugative transfer SAXS Structure of Full-Length NES with DNA. Full-length NES alone was examined in S. aureus, using the staphylococcal conjugative and in complex with oriTHP30 DNA were examined by SAXS for plasmid family prototype pSK41 because its conjugative transfer low-resolution structure determination. NES alone produced genes are nearly identical to pLW1043 but it lacks the dangerous SAXS data with clear concentration-dependent radius of gyra- vancomycin-resistance transposon. Disruption of the nes gene tion (Rg) values, indicating the formation of dynamic complexes in pSK41 led to the elimination of plasmid transfer, as measured not amenable to interpretation. The complex of full-length NES by the number of DNA transfer events (the formation of with DNA was well behaved, however, and gave a concentration- “transconjugants”) per donor S. aureus cell (Fig. 4A). This result independent 38-Å Rg in solution (Fig. S4 A and B). A dummy confirms the essential role of NES in the transmission of pSK41 atom bead model and associated molecular envelope of the and related staphylococcal plasmids, including pLW1043 (14). NES–DNA complex were calculated using MONSA (23) and Importantly, whereas complementation with wild-type nes returns Chimera (24), respectively. These revealed the overall shape of conjugation to wild-type levels, complementation with specific the complex (Fig. 3D, top, pink) and the location of the relatively mutant nes genes designed on the basis of the NES crystal structures electron-rich DNA (Fig. 3D, top, yellow). SASREF (25) was dramatically impacted plasmid transfer (Fig. 4A). Variant nes genes used for rigid-body fitting of the structures of the N-terminal containing the deletion of either loop1 or loop2, both of which relaxase–DNA complex and C-terminal domain crystal struc- significantly disrupted the DNA cleavage-religation equilibrium of tures into this SAXS envelope. A solution was chosen that both NES in vitro (Figs. 2 and 3A), significantly reduced conjugative accommodated the SAXS envelope and placed the DNA of the DNA transfer (Fig. 4A). Thus, the specific major- and minor-groove relaxase complex within the electron-rich region (Fig. 3D, top). DNA contacts formed by NES loops 1 and 2 are essential for In addition, it positioned a positively charged region of the conjugative DNA transfer between S. aureus cells. Furthermore, C-terminal domain in close proximity to the DNA-bound NES the C-terminal domain of NES is also required for conjugation. relaxase active site (Fig. 3D, bottom). Thus, this SAXS-based Introduction of a stop codon after the nucleotides encoding structural model appears to corroborate the functional obser- residue 303 in nes eliminated plasmid transfer in S. aureus (Fig. 4A). vation regarding the impact of the C-terminal domain on NES This residue was chosen because the nes gene in the MRSA plasmid relaxase activity (Fig. 3C). Together, they suggest a putative model pUSA03 is truncated at this position (26), as discussed below.

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254

195

Fig. 3. Functional analysis of NES. (A) NES wild-type (WT) and variant relaxase activities, including that of loop 1 (L1) and loop 2 (L2) deletions. (B) Activity of NES relaxase with variant oligos, including an abasic G26 site. (C) Effect of DNA oligo length on NES relaxase and full-length activity. (D) Upper, SAXS envelope shape (pink) repre- senting averaged and filtered dummy atom model of the NES–DNA complex, with the relatively electron-rich region indicated (yellow) and NES crystal structures docked by SUPCOMB. Lower, a positively charged region of the NES C-terminal domain is in proximity to the DNA-bound active site of the NES relaxase in the docked model.

Taken together, these data establish that the loop 1 and 2 regions this compound was compared with that of a mismatch polyamide 2, of the NES relaxase domain, and the C-terminal domain of NES, which shows lower affinity for the 5′-GCGAA-3′ sequence (Fig. S5). are required for the transfer of plasmids in S. aureus cells. The DNA cleavage activity of the relaxase 1–220 region of NES was eliminated by match polyamide 1 at 50 μM(Fig. S6). Interestingly, Polyamide Inhibition. The dependence of conjugative plasmid 2.5- to 50-μM concentrations of the mismatch control polyamide 2 transfer on an intact DNA-binding domain suggests the potential caused a significant increase in product formation by the relaxase for inhibiting relaxase function and limiting the spread of anti- (Fig. S6). This result is similar to those obtained when the loop 1 and 2 biotic resistance genes through disruption of NES–DNA interac- regions of the relaxase were deleted (Fig. 3A). Taken together, these tions. As a proof-of-concept, we examined the ability of synthetic data indicate that, for the isolated relaxase domain, disrupting the minor-groove binding ligands to inhibit NES activity. The GC-rich protein–DNA interactions around the DNA hairpin can significantly hairpin sequence recognized by NES represents a low-affinity target shift the enzyme’s cleavage-religation equilibrium. This effect, how-

for traditional minor-groove binding of natural products such as ever, is not seen when intact, 1–665 NES is used, as outlined below. BIOCHEMISTRY distamycin and netropsin, which preferentially bind AT tracts With full-length NES, mismatch polyamide 2 showed no effect, (27, 28). Indeed, in our hands we observed no significant change in whereas the match polyamide 1 significantly reduced DNA cleavage NES relaxase activity when netropsin was added up to concen- activity at 25 μM(Fig.4C). A closer examination of NES inhibition by trations of 1 mM. This led us to investigate inhibition of NES by Py- concentrations of polyamide 1 between5and50μM produced in- fi Im polyamides, a class of sequence-speci c minor-groove binding hibition curves and IC50 values of 18 and 21 μM for the full-length compounds that can be programmed to bind GC-rich sequences and relaxase regions of NES, respectively (Fig. 4D). Thus, full-length with high affinity (Fig. 4B)(29).Matchpolyamide1 was designed to NES is inhibited by a polyamide targeted to the hairpin minor-groove bind selectively to the 5′-GCGAA-3′ sequence contacted by the DNA sequence and is resistant to the effects of the nonspecific essential loop 1 of NES (Figs. 1A and 2B). As a control the activity of polyamide. Taken together, these results confirm that full-length

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Fig. 4. NES in S. aureus and polyamide inhibition. (A) Effect of nes deletion (KO) and complementation with WT and designed NES loop 1 (L1), loop 2 (L2), and C-terminal deletion (ΔC-term) mutants on conjugation in S. aureus.(B) Structures of the Match Polyamide 1 and Mismatch Polyamide 2.(C) Inhibition of

full-length NES with Match 1 (solid bars) and Mismatch 2 (shaded bars) polyamides. (D) Eighteen- and 21-μMIC50 values of the Match Polyamide 1 for the full- length and relaxase forms of NES, respectively.

NES acts in a manner distinct from that of the isolated relaxase with Minor-groove–targeted Py-Im Polyamide 1, designed to bind respect to catalytic activity and suggest that with further development to the GC-rich DNA minor groove of the NES substrate hair- small molecules capable of inhibiting NES–DNA interactions could pin, demonstrated IC50 values of 20 μMwithrespecttoDNA disrupt NES function to prevent antibiotic resistance transfer. cleavage. Although optimization of polyamide architecture and functionalization will be necessary to promote organism-spe- Discussion cificuptakeinS. aureus, these results suggest a future approach Antibiotic resistance propagation is largely mediated by the for targeted disruption of antibiotic resistance transfer. Minor- conjugative DNA transfer of plasmids encoding resistance genes, groove binders similar to Py-Im polyamides have shown activity as well as nearly all of the factors required for DNA transmission against S. aureus in vitro and in animal models of infection (31, from donor to recipient cells (7, 9, 30). We focused on the NES 32). Interestingly, there is a natural precedent for targeting NES relaxase enzyme from plasmid pLW1043, the first vancomycin- activity as a means to limit DNA transfer, in the form of a func- resistance vector identified in S. aureus (4). pLW1043 and related tional CRISPR containing a nes-specific spacer that is present in plasmids like pSK41 variously harbor resistance to key antibiotics the genome of Staphylococcus epidermidis strain RP62a (33). like vancomycin, aminoglycosides, β-lactams, and mupirocin (6). This is a unique single-tyrosine relaxase structure resolved in The NES relaxase was shown to contain two essential loops (1, 2) complex with DNA, and DNA binding by NES is clearly distinct that close around the bound DNA hairpin (Figs. 1A and 2). We relative to the “multityrosine” relaxase–DNA complex structures establish that the disruption of these loops, using either muta- elucidated previously. The cavity that holds the buried Gua-26 genesis or a polyamide, inhibits NES activity in vitro (Figs. 3A nucleotide base in NES is conserved in the single-tyrosine and 4 B–D). Furthermore, the deletion of either of these loops relaxase structure of plasmid R1162 MobA determined without eliminates plasmid DNA transfer in S. aureus (Fig. 4A). DNA (Fig. S7A and Fig. 2D) (15). In contrast, the multityrosine

2808 | www.pnas.org/cgi/doi/10.1073/pnas.1219701110 Edwards et al. Downloaded by guest on September 26, 2021 relaxases from plasmids pCU1, R388, and F all position a helix in and the C-terminal domain, respectively (Table S1), were collected at the place of the short loop in NES (Fig. S7 B–D) (16–18), and these National Institute of General Medical Sciences and National Cancer Institute helices occlude the Gua-26 binding pocket. Thus, whereas the Collaborative Access Team (GM/CA-CAT). Structures were determined and multityrosine relaxases use a “thumb” region not present in NES refined using Phenix and manually adjusted with Coot (SI Methods). or MobA (Fig. S7 C and D), the shorter single-tyrosine relaxases likely rely on burying a nucleotide adjacent to its active site to DNA-Binding Studies. Direct binding to relaxase and full-length constructs align the substrate DNA. Recall that deletion of Gua-26 signifi- were measured using fluorescence polarization anisotropy and a 6FAM- cantly impacted the equilibrium cleavage activity of NES (Fig. 3B). oriT31 (Table S3). Competition binding studies used fluorescence polariza- ThesedatasuggesttheGua-26binding pocket in NES-like relaxases tion anisotropy and titrations of unlabeled DNA (SI Methods). may be targeted in future studies to disrupt enzyme activity. The structural and biochemical data presented here, together DNA Cleavage Assays. Equilibrium DNA cleavage was examined using 6FAM- with in vivo observation that the deletion of the NES C-terminal labeled oligos with product formation monitored on PAGE gels. Percentage domain significantly reduces conjugation, suggest that the C- of product formation was quantified using samples in triplicate (SI Methods). terminal domain regulates the NES processing of the DNA sub- strate during conjugative transfer. The 11 deposited S. aureus nes Small-Angle X-ray Scattering. Full-length NES was examined in complex with genes are identical in amino acid sequence within their conserved oriTHP30 DNA, using SAXS on a Rigaku Bio-SAXS 1000 instrument with 60- N-terminal relaxase domains (residues 1–220). In addition, 7 en- min collection scans at three concentrations. Concentration-independent fi code 665-residue proteins with complete C-terminal domains. In Rg values were obtained, and averaged and ltered dummy atom models contrast, 4 exhibit significantly shorter C-terminal regions, in- were created and used for docking (25) (SI Methods). cluding one truncated at residue 555 (accession no. EIK19451) and another that ends at position 482 (ADM29113) (34) (Fig. S8). Conjugation Assays in S. Aureus. An nes gene knockout S. aureus strain was The 2 remaining sequenced S. aureus nes genes have severely generated using the Lactococcus lactis Ll.LtrB group II intron (35). WT and limited C-terminal domains, terminating their coding regions at mutant nes complementation plasmids were generated in pSK5632 in which residues 300 or 303 (CCJ09841, YP_492718) (Fig. S8). The 303- nes transcription was driven by a lac promoter (36). Matings were performed residue NES (YP_492718) is produced by a frameshift in the nes on solid surfaces for 20 h, followed by cell recovery and plating with anti- gene on plasmid pUSA03 in some USA300 strains of MRSA (26). biotic selection to determine conjugation frequency (SI Methods). The truncated NES we used was created to mimic this 303-residue protein. Our finding that the C-terminal 362 residues of NES were Polyamide Inhibition Studies. Polyamides were synthesized and characterized, required for efficient conjugation strongly suggests that pUSA03 and their effects on full-length and relaxase domain NES constructs were plasmids with truncated nes genes would be unable to conjugate on determined as described in SI Methods. their own and may require factors present on coresident plasmids. ACKNOWLEDGMENTS. This work was supported by National Institutes of Methods Health Grants AI078924 (to M.R.R.) and GM51747 (to P.B.D.), by National Medical Research Council of Australia Grant 457454 (to S.M.K. and N.F.), by Crystal Structure Determination. Purified NES constructs were crystallized American Cancer Society Fellowship PF-10-015-01-CDD (to J.L.M.), and by using hanging drop vapor diffusion, and data for the relaxase–DNA complex the APS and GM/CA CAT.

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